The initial contact site of the CD4 receptor on pre-fusion, closed HIV-1 envelope (Env) trimer is highly conserved, and is targeted by neutralizing antibodies. This site is an important target for vaccine and therapeutics development. Structural definition of this initial interaction has been a challenge because conformational changes in the HIV-1 Env trimer follow immediately upon CD4 engagement. There are no high resolution structures of the initial contact of the pre-fusion, closed HIV-1 Env trimer with CD4. The structural details of the conformational changes that follow immediately upon CD4 binding are also not known. Precise definition of initial CD4 contacts with the closed HIV-1 Env trimer will fill a gap in our understanding of this early event in HIV-1 entry, and will provide atomic level information for structure-based immunogen design. The overall goals of this study, therefore are, (i) to define, at atomic level details, the initial site of CD4 binding on pre-fusion, closed HIV-1 Env trimer, (ii) to define the steps leading to CD4-induced protomer opening, and (iii) to elucidate CD4-mediated changes in the HIV-1 fusion peptide, an immunodominant region critical for mediating HIV-1 CD4+ T cell entry, and itself a target of neutralizing antibodies. The scientific premise of this grant is that the initial contact of CD4 with HIV-1 Env is the critical first step that determines virus attachment. Although CD4 can bind to multiple Env conformations, this first site of contact on the HIV-1 Env trimer is the target of broadly neutralizing antibodies and effective drugs, hence high resolution structural details of this interaction and a mechanistic understanding of subsequent conformational changes will facilitate the development of intervention strategies that include immunogen design for HIV-1 vaccine efforts and drug design for novel cure AIDS strategies to eliminate the latent pool of HIV-1-infected CD4 T cells. The innovation in this grant derives from advances in cryo-EM technology that include new grid preparation and specimen vitrification methods, improved microscope hardware, automated methods for high-throughput data collection, and advanced algorithms for data processing. These advances have recently allowed us to establish a rapid pipeline for determining high resolution structures of HIV-1 Env complexes. The innovation also derives from availability of panels of native-like Env constructs and antibodies for the proposed structural and mechanistic analyses. At the completion of this study we expect to provide a movie for CD4-induced opening of the HIV-1 Env trimer. High resolution structures of the initial CD4 contact on the closed HIV-1 Env trimer will provide atomic level information for structure-based immunogen and drug design. Visualization of the initial steps of CD4-induced Env opening will provide information on which Env regions move first, and will inform the design of stabilized immunogens. This study will also provide an understanding of the CD4-induced conformational diversity sampled at the HIV-1 fusion peptide region and how antibodies, both natural and vaccine-elicited, respond to it.